Absence of SIV-specific CD8+ T cells in germinal centers may set the stage for persistent infection

CD8+ T cells play an important role in controlling of HIV and SIV infections. However, these cells are largely excluded from B cell follicles where HIV and SIV producing cells concentrate during chronic infection. It is not known, however, if antigen-specific CD8+ T cells are excluded gradually as pathogenesis progresses from early to chronic phase, or this phenomenon occurs from the beginning infection. In this study we determined that SIV-specific CD8+ T cells were largely excluded from follicles during early infection, we also found that within follicles, they were entirely absent in 60% of the germinal centers (GCs) examined. Furthermore, levels of SIV-specific CD8+ T cells in follicular but not extrafollicular areas significantly correlated inversely with levels of viral RNA+ cells. In addition, subsets of follicular SIV-specific CD8+ T cells were activated and proliferating and expressed the cytolytic protein perforin. These studies suggest that a paucity of SIV-specific CD8+ T cells in follicles and complete absence within GCs during early infection may set the stage for the establishment of persistent chronic infection. Author Summary A paucity of SIV-specific CD8+ T cells in lymphoid follicles and complete absence within most follicular germinal centers during early infection may set the stage for the establishment of persistent chronic infection.


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We further compared the percentage of SIV-specific tetramer+ CD8+ T cells that express PD-1 between 137 early and our previously published data (Li et al., 2016) from chronic SIV infection in follicular and 138 extrafollicular regions respectively. Again, no significant differences were observed ( Fig. 3C and 3D).

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Regulatory T cells (Tregs) play a crucial role in maintaining immunological self-tolerance and   of Foxp3+ SIV-specific tetramer+ CD8+ T cells inside and outside B cell follicles also showed no 158 significant difference (Fig. 4D).

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Similar to SIV-specific CD8+ T cells, Foxp3+ cells levels were also significantly lower in 160 follicular than extrafollicular regions ( Fig. 4E and 4F). In addition, there was no significant difference 161 between the ratios of Tet+ cells: Foxp3+ cells in follicular and extrafollicular regions (data not shown).

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These findings suggest that contact mediated suppression of Foxp3+ Tregs on SIV-specific CD8+ T cells 163 is similar in follicular and extrafollicular regions in early SIV infection.

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We next evaluated whether the relationship of Foxp3+ Tregs and SIV-specific CD8+ T cells

Cell death is not likely mediating low levels of follicular SIV-specific CD8+ T cells 207
A hypothesis yet to be tested regarding the relative low abundance of virus-specific CD8+ T cells 208 in follicles is that follicular virus-specific CD8+ T cells die via apoptosis at greater rates than 209 extrafollicular virus-specific CD8+ T cells. Here we tested that hypothesis and determined the levels of  In addition to determining the quantity of SIV-specific CD8+ T cells in follicular and 252 extrafollicular compartment of lymph nodes during early infection, we also determined the phenotype of these cells. We found a broad range of SIV-specific CD8+ T cells expressing PD-1 during early infection.

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The PD-1 expression on SIV-specific CD8+ T cells during early infection likely reflects cells that were 255 recently were exposed to antigenic stimulation, however, exhaustion cannot be ruled out. We found

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In this study, we also found similar levels of SIV-specific CD8+ T cells in follicular and 278 extrafollicular areas of lymph nodes expressing the apoptosis marker PARP. These findings suggest that 279 the relative low abundance of virus-specific CD8+ T cells in follicular relative to extrafollicular regions 280 of lymph nodes is not likely due to follicular virus-specific CD8+ T cells dying via apoptosis at increased 281 rates compared to extrafollicular virus-specific CD8+ T cells. perforin expression, tetramer + cells were scored using the following objective criteria as follows.

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Tetramer + cells with no detectable perforin staining above background levels were scored as perforin 337 negative. Tetramer + cells with perforin staining 2-3X greater than background were scored as perforin low,

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with perforin staining 4-9X higher than background as perforin medium, and those with 10X or greater 339 than background levels and with perforin staining detectable throughout much of the cytoplasm were 340 scored as perforin high. Cell counts were done on single z-scans. While doing the cells counts, we 341 demarcated cells using a software tool to avoid counting the same cell twice. All quantitative image 342 analyses were done with lymph node tissues. An average of 7.42 mm 2 (range, 5.63-10.08 mm 2 ) was 343 analyzed for each lymph node.

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In situ hybridization combined with immunohistochemistry

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In situ hybridization for SIV RNA was performed as previously described (Connick et al., 2014).

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This technique identifies cells that are actively transcribing SIV, but not extracellular virions encapsulated